Project Summary Tissue resident macrophages function as essential components of the innate immune system by serving as sensors and responders to infection and injury. Functional and transcriptomic studies further indicate that macrophages residing within different tissues are phenotypically distinct and exhibit correspondingly different programs of gene expression that enable tissue-specific functions. In addition to their immune and homeostatic functions, resident macrophages and infiltrating monocyte-derived cells also contribute to a diverse array of metabolic and degenerative human diseases. Studies performed during the last funding cycle of this grant demonstrated that different tissue environments play instructive roles in promoting distinct macrophage phenotypes by driving the selection and function of cell specific enhancers. In parallel, studies supported by this grant leveraged the effects of non-coding natural genetic variation provided by five different strains of mice to investigate mechanisms controlling bone marrow-derived macrophage specific gene expression in vitro. Evaluation of the effects of >50 million SNPs and InDels provided evidence for roles of ~100 TFs in shaping lineage-determining factor binding and gene expression. Here, we propose to advance these genomic and genetic approaches to identify mechanisms that specify the molecular identities of the resident and recruited macrophages of the mouse and human liver in health and metabolic disease. Specific Aim 1 will test the hypothesis that local DLL4, BMP/TGFb and desmosterol function in a sequential and combinatorial manner to drive the selection and activation of Kupffer cell-specific enhancers by regulating RBPJ, SMADS and LXRs, respectively. Specific Aim 2 will define the network of collaborative transcription factors required for establishing the Kupffer cell enhancer landscape and quantify cell autonomous and non-cell autonomous effects of natural genetic variation. Specific Aim 3 will investigate gene by environment interactions that regulate myeloid cell phenotypes in NASH. Experimental strategies developed in Specific Aims 1 and 2 will be used to test the hypothesis that myeloid diversity is the consequence of microenvironment-specific combinations of signals that differentially reprogram the enhancer landscapes of resident Kupffer cells and recruited macrophages. These studies are expected to lead to the identification of disease- and niche-specific signaling pathways that contribute to pathological macrophage phenotypes in NASH. Specific Aim 4 will define the transcriptomes and epigenetic landscapes of myeloid populations in the healthy human liver and across the spectrum of non-alcoholic fatty liver disease. These studies will establish similarities and differences of human and mouse Kupffer cells, define effects of genetic variation across individuals, and provide a map of regulatory landscapes of these cells that can be used for interpretation of non-coding GWAS risk variants.